Influence of the Chitosan Surface Profile on the Nucleation and


Sukun Zhang† and Kenneth E. Gonsalves*. Polymer Program at the Institute of Materials Science & Department of Chemistry,. University of Connecticut,...
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Langmuir 1998, 14, 6761-6766

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Influence of the Chitosan Surface Profile on the Nucleation and Growth of Calcium Carbonate Films Sukun Zhang† and Kenneth E. Gonsalves* Polymer Program at the Institute of Materials Science & Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 Received August 26, 1997. In Final Form: August 5, 1998 Calcium carbonate crystal growth on a chitosan substrate was achieved using a supersaturated calcium carbonate solution. Heterogeneous nucleation of the calcium carbonate crystals was significantly influenced by the chitosan surface profile. Poly(acrylic acid) (MW ) 2000, PAA2K) was chosen to modify chitosan surfaces in aqueous solution. The adsorption of PAA2K occurred on the chitosan film surface. The thicknesses of the adsorbed chitosan films were determined by ellipsometry. X-ray photoelectron spectroscopy (XPS) was used to determine the surface structure of the modified chitosan films. Calcium carbonate crystal sizes and morphology were observed under a polarized optical microscope and a scanning electron microscope (SEM). PAA2K promoted the heterogeneous nucleation of calcium carbonate crystals on the chitosan film and at the same time suppressed homogeneous nucleation in solution.

Introduction In nature, biological organisms produce polymerinorganic hybrids, such as structural materials (shell, bone, and wood) and functional materials [otoconia (gravity devices), trilobites (optical lenses), and coccoliths (protection)].1 Calcium salts are the major inorganic materials in these systems. Small amounts of acidic-rich proteins play a major role in forming these hybrids by influencing the mineral crystal nucleation and growth.2 These hybrids have superior mechanical properties as compared to synthetic hybrids.3 Recently, the synthesis of inorganic-organic hybrids by mimicking biomineralization has attracted great attention; however, only discrete mineral crystals have been obtained on organic matrixes.4,5 Little has been achieved in fabricating complex, dense films. Several factors could influence mineral nucleation and crystal growth on a polymer film, such as the degree of saturation of a supersaturated solution and the surface charges on a polymer film. It is very crucial for successful biomimetic synthesis that only heterogeneous nucleation be promoted on the polymer film and that homogeneous nucleation be suppressed in the mother liquid. Heterogeneous nucleation forms strong bonds at the interface between a substrate and mineral crystals, which promotes a strong interface between the polymer and ceramics.6 A high degree of saturation of the mother solution favors homogeneous nucleation from solution.7 On the other hand, strong electrostatic interaction, polar acid-base interaction, and stereochemical recognition between inorganic † Current address: Westvaco Research Center, 5600 Virginia Ave., Charleston, SC 29423.

(1) Lee, S. L.; Veis, A.; Glonek, T. Biochemistry 1977, 16, 29712978. (2) Weiner, S.; Traub, W. FASEB J. 1992, 6, 879-885. (3) Wade, V. J.; Levi, S.; Arosio, P.; Treffry, A.; Harrison, P. M.; Mann, S. J. Mol. Biol. 1991, 221, 1443-1452. (4) Mann, S. Nature 1988, 332, 119-124. Mann, S. Chem. Mater. 1993, 6, 311. (5) Calvert, P. Mater. Sci. Eng. 1994, C1, 69. (6) Bunker, B. C.; Rieke, P. C.; Tarasevich, B. J.; Campbell, A. A.; Fryxell, G. E.; Graff, G. L.; Song, L.; Liu, J.; Virden, J. W.; McVay, G. L. Science 1994, 246, 48-55. (7) Ebrahimpour, A.; Perez, L.; Nancollas, G. H. Langmuir 1991, 7, 577-583.

crystals and an organic matrix favor the heterogeneous nucleation of mineral crystals. Therefore, one key factor for successfully mimicking biomineralization to coat a dense ceramic film on organic polymer substrates is to increase the density of charges or polarity on the substrate surface. In this paper, we describe the influence of a modified chitosan surface profile on calcium carbonate crystal nucleation and growth. Experimental Section Materials. Chitosan was obtained from Sigma Chemical Co. Poly(acrylic acid) (PAA) with MW ) 2000 (PAA2K) was purchased from Aldrich. Calcium carbonate calcite crystals were obtained from the Geology Department of the University of Connecticut, and calcium carbonate calcite powder was purchased from Sigma. Glass slides, silicon wafer, and poly(methyl methacrylate) (PMMA) were used as supports to make chitosan films. Sample Preparation. Preparation of Calcium Carbonate Supersaturated Solution. Supersaturated calcium bicarbonate solution was prepared following the procedure described by Kitano.8 A suspension of CaCO3 (calcite) with the ratio 0.9/100 (mg/mL) of calcium carbonate to distilled water was first placed in a three-neck, round-bottomed flask. The flask was equipped with a gas inlet and an outlet on its side and center sockets, respectively. A glass stopper was also placed in the third neck. A magnetic stirrer was used. While the solution was stirred, carbon dioxide was bubbled into the system at room temperature for 6 h. The suspension was then filtered, and the filtrate was purged with carbon dioxide gas again for 30 min to dissolve any remaining crystals. The pH value of the resulting supersaturated solution was about 6.00. PAA was first dissolved in water, and then a small amount of the solution was injected with a microsyringe into a polystyrene (PS) bottle, which contained a chitosan film. The additive aqueous solution was added before pouring the calcium carbonate supersaturated solution into the container. Preparation of Chitosan Film. A chitosan film was obtained by casting the 1% (w/w) solution of chitosan in a 1% (w/w) acetic acid aqueous solution on a PMMA plate, glass slide, or silicon wafer. The glass slides and silicon wafers were treated in a chromic acid-sulfuric acid cleaning agent overnight and then repeatedly rinsed with water. The slides and wafers were again rinsed three times with filtered acetone before use. In a typical preparation of the casting solution, 2 g of chitosan was added to 100 mL of distilled water and the resulting solution was stirred (8) Kitano, Y. Bull. Chem. Soc. Jpn. 1962, 12, 1980.

10.1021/la970962s CCC: $15.00 © 1998 American Chemical Society Published on Web 10/22/1998

6762 Langmuir, Vol. 14, No. 23, 1998 for 10 min. Then 100 g of a 2% (w/w) acetic acid aqueous solution was added, and the resulting solution was stirred at room temperature for another 30 min.9 A viscous solution was obtained and filtered. The chitosan film made in this manner was then neutralized with dilute ammonium hydroxide (5 wt %) and washed extensively with water. It was dried at room temperature. Characterization. X-ray photoelectron spectroscopy (XPS). XPS was used to determine the surface structure of the chitosan film. The spectra were collected on a monochromatic spectrometer (Perkin-Elmer, PHI 5300) using an Al KR source (600 W, 15 keV) with pass energies 37.5 eV for utility scans and 89.45 eV for survey scans. A vacuum was maintained at approximately 10-9 Torr. Survey scans were used to ensure that the elemental composition at the surface was as expected. The higher resolution utility scans were used to determine the atomic concentrations of carbon, oxygen, nitrogen, and calcium. XPS can also provide chemical shift information. Typically, a sample for XPS measurement was prepared by spin coating the chitosan solution on a silicon wafer. The chitosan films adsorbed with PAA2K were removed from solution and rinsed carefully with distilled and deionized water. The prepared samples were then introduced into the XPS sample chamber within 6 h. Ellipsometry. The thicknesses of the adsorbed chitosan films were determined by ellipsometry under the assumption that chitosan and PAA have the same reflective index. PAA was adsorbed onto the chitosan film from a PAA aqueous solution or from the supersaturated calcium carbonate solution with different concentrations of PAA, under the same conditions as those utilized for the crystal growth. Chitosan films were spin-coated on silicon wafers. The dried films were measured before and after soaking in the solutions in a clean room. The amount of adsorbed PAA was obtained by subtracting the original film thickness from the adsorbed film thickness. Three angles were chosen for the experiment, 60°, 70°, and 80°. The wavelength was changed from 300 to 1000 nm in steps of 25 nm. pH Measurement. The pH of a solution was measured using a Fisher pH meter (model 107). The probe of the pH meter was soaked in distilled and deionized water overnight before measurement. The probe was rinsed five times before each measurement. Care was required to avoid introduction of impurities into the crystallization system. Optical Microscope (OM) and Scanning Electronic Microscope (SEM). CaCO3 crystal sizes and morphology were observed under a polarized optical microscope using a Nikon model Labophotpol and also by SEM (AMR 1200B). Birefringence was observed using polarized light with a 1-L accessory plate. The prepared chitosan-calcium carbonate hybrid film was observed under the optical microscope. Only smooth samples could be observed under the OM. For hybrids obtained from the thick chitosan film (those obtained from casting film on a PMMA plate), it was not possible to make an observation because of the twist of the film after crystal growth. SEM was employed for such hybrids. A gold layer was coated before the hybrids were introduced into the SEM sample chamber.

Results and Discussion Adsorption of PAA2K on Chitosan Film. The adsorption of PAA (MW ) 2000, PAA2K) onto chitosan films was determined using an ellipsometer. The threelayer model was used (Zhang, S. Ph.D. Dissertation, University of Connecticut, Storrs, CT, 1996), and the film thickness was measured as a function of wavelength at different concentrations of PAA2K. The adsorbed layer thickness as a function of PAA2K concentration in aqueous solution is shown in Figure 1. As the concentration of PAA2K increased, the thickness of the adsorbed layer increased rapidly and leveled off above 600 ppm. On further increase of the PAA2K concentration, the thickness of the adsorbed layer of PAA on the chitosan film did not increase. (9) Austin, P. R.; Willmington, D. U.S. Patent 4,309,534, 1982.

Zhang and Gonsalves

Figure 1. Adsorbed thickness of PAA2K onto chitosan film from PAA2K aqueous solution.

Figure 2. Adsorption behavior of PAA2K on chitosan films from aqueous solution at different concentrations of PAA2K.

The adsorption behavior of PAA2K from aqueous solution onto chitosan films at various concentrations of PAA2K is shown in Figure 2, where the adsorbed layer thickness as a function of time is plotted. At a low concentration of PAA2K (25 ppm) with the pH of the solution at 5.82, the adsorbed layer thickness of PAA2K on the chitosan film was low, and a long time was required to reach equilibrium. The adsorbed layer thickness was much higher and equilibrium was reached more quickly when the concentration of PAA2K was higher. The chitosan film swelled more at pH 2.48, as the chitosanfilm surface was mainly in the form of polycations. Therefore, the rate of the PAA2K penetration into chitosan film was faster, and more surface area became available for adsorption. The degrees of dissociation (R) of both the carboxylic groups in PAA and amine groups in chitosan in aqueous solution depend on the pH of the solution. As a consequence, both the surface charge density of the chitosan film and PAA in solution changed. The dissociation constant pKa is 4.5 and 6.3 for PAA and chitosan, respectively.10 Therefore at high pH (pH > 6.3), PAA is (10) Chibowski, S. J. Colloid. Interface Sci. 1990, 140, 444.

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Figure 4. Adsorption behavior from the supersaturated calcium carbonate solution and aqueous solution.

Figure 3. Scheme of existing forms of poly(acrylic acid) (PAA2K) and chitosan in aqueous solution. Table 1. pH Values of Solutions at Different Concentrations of PAA2K solution aqueous solution

supersaturated calcium carbonate solution (4 h)

PAA concn (ppm)

pH

25 200 500 750 1500 2000 50 200 2000

5.82 4.10 2.85 2.56 2.56 2.48 6.30 6.40 5.25

in the form of polyanionic chains in solution, while the chitosan film is close to a neutral polymer, as shown in Figure 3. In contrast, at low pH (pH < 4.5), the chitosanfilm surface is in the form of polycations, and PAA is close to a neutral polymer. The pH value of the aqueous solution dropped with an increase in the concentration of PAA2K. However, the pH in a supersaturated calcium carbonate solution changed only slightly as the concentration of PAA2K changed from 25 to 2000 ppm (Table 1). At low concentration, both in an aqueous solution and in a supersaturated calcium carbonate solution, PAA2K is completely dissociated in the form of polyanionic chains in the solutions (pH > 5.33). At concentrations of 25 and 200 ppm, the adsorbed layer thicknesses from water and from a supersaturated calcium carbonate solution were similar (Figure 4). At a high concentration of PAA2K (2000 ppm), the adsorbed layer thickness from aqueous solution was greater than that from a supersaturated calcium carbonate solution. In aqueous solution, the pH dropped quickly to 2.56 as the concentration of PAA2K was raised to 2000 ppm. At this pH, PAA was in the form of polyacid; thus, the adsorption behavior was close to that of neutral polymer chains. As a result, a thick adsorbed layer was obtained. In the case of a supersaturated calcium carbonate solution with 2000 ppm PAA2K, on the other hand, the adsorbed layer thickness was much lower than

that from the same concentration of PAA2K in an aqueous solution. The pH reduction was smaller compared to that for the aqueous solution, as can be seen in Table 1. For a PAA2K concentration of 200 ppm, the adsorbed layer thickness from a supersaturated solution was slightly higher than that from an aqueous solution (Figure 4). However, the pH values of these two systems differed, with a lower pH for the aqueous solution. Thus, some of the PAA chains were not dissociated at this pH value and existed in the form of polyacid. In this case, the adsorbed layer thickness should have been higher because of the weak repulsion between PAA2K chains. However, the experimental result showed the opposite result here. The only difference between the two systems was the existence of ions in the supersaturated solution. The ionic strength was 0.02 M, which is low. According to theory,11 for oppositely charged surfaces which adsorb polyelectrolyte, the adsorption of a weak polyelectrolyte on a highly oppositely charged surface depends only weakly on ionic strength when the latter is low. Therefore, the measured adsorption layer from the supersaturated solution was thicker than expected because of the existence of calcium carbonate nuclei on the chitosan-film surface. Therefore, the absorbed layer thickness from a calcium carbonate solution was slightly higher than that from an aqueous solution. More calcium carbonate nuclei may be deposited on the chitosan-film surface for a PAA2K concentration of 200 ppm than for PAA2K 25 and 2000 ppm as determined by XPS and discussed in detail below. XPS Analysis of Chitosan-Film Surfaces. XPS can be used as a more direct probe of the surface composition profile. Chitosan thin films, spin coated on silica wafer, were soaked in a supersaturated solution for 4 h with or without PAA2K and monitored at a takeoff angle of 62°. XPS survey scan spectra are shown in Figure 5. In the control sample (without PAA2K), no calcium element was detected. The absence of calcium indicated that no calcium carbonate nuclei were formed at the beginning of the crystallization and also showed that no calcium ions were trapped on the chitosan-film surface. As a result, local saturation of calcium carbonate could not be achieved. The presence of a calcium peak at a binding energy of 350 eV was readily noted when PAA2K was introduced into the crystallization systems. The appearance of calcium in the XPS spectrum resulted from calcium ions which (11) Evers, O. A.; Fleer, G. J.; Scheutjens, J. M. H. M.; Lyklema, J. J. Colloid Interface Sci. 1986, 111, 446.

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Table 2. Atomic Concentrations (AC) of Elements and the Repeat-Unit Ratios of Chitosan to Poly(acrylic acid) on the Chitosan-Film Surface Soaked in a Supersaturated Solution at the Different Concentrations of Poly(acrylic acid) for 4 h conc of PAA2K (ppm)

C (AC %)

O (AC %)

N (AC %)

Ca (AC %)

N/N+ (AC/AC)

PAA/chitosan (repeat unit ratio)

0.0 100 200 600 1000

55.4 56.3 57.8 56.9 59.1

36.3 38.0 37.2 38.3 37.4

8.3 4.6 4.1 3.7 3.5

0 1 0.97 1.18

95/5 50/50 62/38 51/49 50/50

0.0 2.62 2.50 3.23 3.28

Figure 5. XPS survey scan spectra of chitosan-film surfaces soaked in a calcium carbonate supersaturated solution with different concentrations of PAA2K for 4 h.

were trapped on the chitosan film or from nucleation of calcium carbonate crystals in the presence of PAA. In this case, both positive and negative charges occurred on the chitosan film. Table 2 summarizes both the atomic concentrations of elements and the repeat-unit ratios of chitosan to PAA on the chitosan-film surface soaked in a supersaturated solution at different concentrations of PAA2K for 4 h. The atomic concentration ratios of free nitrogen to protonated nitrogen were around 1:1 after addition of PAA2K into the systems. The atomic concentration of nitrogen decreased dramatically after addition of PAA2K to the system and further decreased slightly as the concentration of the PAA2K increased, while the repeat-unit ratio of PAA to chitosan showed the opposite trend. Therefore, the higher the concentration of PAA2K, the more PAA2K was adsorbed onto the chitosan-film surface. Above 600 ppm, the adsorption reached equilibrium. At the concentration of 1000 ppm, greater numbers of mobile carboxylate anions of PAA in the solution inhibited crystallization. Angle-dependent XPS also provides a means of obtaining a surface composition profile. Each chitosan film with adsorbed PAA2K from aqueous solution was examined at takeoff angles from 15 to 62°. A takeoff angle of 90°

represents the deepest penetration probe into a material, while a takeoff angle of 0° would be the most sensitive to surface conditions. The results of angle-dependent XPS at various concentrations of PAA2K in aqueous solution are summarized in Table 3. As can be seen, the surfaces were enriched in polar elements, for instance, oxygen and nitrogen, when the concentrations of PAA2K were 200 and 2000 ppm. The enrichment decayed as the probe depth increased. An opposite profile was observed for the sample obtained from a 25 ppm PAA2K solution. Since the nitrogen only existed in chitosan and not in PAA, the atomic concentration of nitrogen can indicate the surface coverage by PAA2K. Figure 6 gives the nitrogen concentration of chitosan film soaked in aqueous solution with various contents of PAA2K. The high nitrogen concentration occurred at the low content of PAA2K, i.e., 25 ppm, which indicated the low coverage of PAA2K on the chitosan film. This was in agreement with the adsorption result determined by ellipsometry, as discussed earlier. In the cases of 25 and 200 ppm PAA2K concentrations, the nitrogen concentration remained the same at different probe depths. An interesting surface profile was observed in the case of PAA content 2000 ppm. A high nitrogen concentration was at the outermost surface layer. The minimum value was at the probe depth where sin(θ) ) 0.5. The concentration increased again as the probe depth increased [increasing in sin(θ)]. The high nitrogen content on the outermost surface was caused by the high ratio of the protonated nitrogen to the amino nitrogen on the chitosan surface. The highly charged nitrogen was most likely to migrate onto the chitosanfilm surface because of the affinity to the polar solvent, water. This observation again indicated that PAA2K short chains not only were adsorbed on the chitosan-film surface but also penetrated into the film. The adsorption experimental showed that the adsorbed layer was in the range of 10-70 nm, while XPS can only penetrate to 20 nm in depth. The observation of nitrogen on the chitosan film determined by XPS indicated the existence of the penetration. The calcium concentration as a function of the sine of the takeoff angle for the chitosan films at various concentrations of PAA2K supersaturated solution is given in Figure 7. The calcium concentration was lower at smaller probe depths, increased with an increase of the sine of the takeoff angle up to sine equal to 0.5, and then leveled off at a plateau value. The same trend occurred in all samples for three different concentrations of PAA2K in a supersaturated solution. The low calcium concentration at the outermost surface was probably caused by the samples being rinsed carefully with water after being picked up from supersaturated solutions. As a consequence, some of calcium adsorbed on the outermost surface was redissolved in water. It was interesting that the calcium concentrations were low at both high and low contents of PAA2K, such as 25 and 2000 ppm. In the case of 200 ppm of PAA2K in the supersaturated solution, the calcium atomic concentration was much higher. This was attributed to the adsorbed amount of PAA2K on the chitosan film being higher in the case of 200 ppm PAA

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Table 3. Atomic Concentration of Carbon, Oxygen, and Nitrogen at Various Takeoff Angles Determined by XPS in Different Concentrations of Poly(acrylic acid) (MW ) 2000) O% equ 1.5m 5s

O%

N%

conc of PPA (ppm)

15°

30°

45°

62°

15°

30°

45°

62°

15°

30°

45°

62°

25 200 2000 200 2000

49.5 48.95 55.4 47.43 50.19

48.2 54.44 60.5 54.50 56.76

49.7 50.95 54.78 52.57 55.23

50.3 50.06 54.88 51.56 57.22

44.9 47.15 41.4 48.02 45.71

45.8 41.89 37.3 40.26 40.26

44.9 45.32 42.41 42.02 41.70

43.9 46.07 41.98 42.90 39.64

5.56 3.90 3.23 4.55 4.11

6.07 3.68 2.23 5.24 2.98

5.45 3.72 2.80 5.41 3.07

5.82 3.87 3.14 5.54 3.14

Figure 6. Atomic concentration of nitrogen as a function of the takeoff angle at different concentrations of PAA2K.

Figure 8. pH of a supersaturated calcium carbonate solution as functions of crystallization time at different concentrations of PAA2K (20 °C).

Figure 7. Atomic concentrations of calcium as a function of the takeoff angle with different concentrations of PAA2K.

than in the case of 25 ppm, as discussed earlier. Since the nitrogen concentration was higher at 25 ppm than at 200 ppm and the ratio of NH3+/NH2 was the same in the two cases, the absolute amount of NH3+ was higher at 25 ppm. In addition, the adsorbed layer thickness in this case was lower than that in the case of 200 ppm, as determined using ellipsometry. Therefore, the amount of carboxylate ions on the chitosan film was low; furthermore, some of the carboxylate ions combined with NH3+ to form a complex, which reduced the ability of carboxylic groups to attract calcium ions. Consequently, the local degree of saturation was low. On the other hand, in the case of 200 ppm PAA2K in the supersaturated solution, the adsorbed amount of carboxylate anions on the chitosan film was higher and few of them formed a complex. As a consequence, the electrostatic interaction between calcium cations and carboxylate anions was strong and caused a local high degree of saturation around the chitosan film, as was determined by XPS. In the case of 2000 ppm PAA2K in the supersaturated solution, the amount of PAA mobile chains in the solution

was much higher as mentioned earlier. As a consequence, the high degree of local saturation would not be generated around the chitosan film. This resulted in the low calcium concentration on the chitosan film. The attenuated total reflection (ATR) spectra of chitosan-film surfaces with and without PAA2K also indicated the existence of complex NH3+ COO-.12 Crystallization of Calcium Carbonate. Crystallization of calcium carbonate was controlled by the following equilibrium in which carbon dioxide was continually lost:

CaCO3(s) + CO2(g) + H2O(l) f 2HCO3-(aq) + Ca2+(aq) On the evolution of carbon dioxide gas from a supersaturated solution, the pH value of the solution increased. The pH value can be used as an indicator to monitor crystallization. Figure 8 shows the pH values of the supersaturated calcium carbonate solution as a function of crystallization time. The pH values of the solutions increased as crystallization proceeded and leveled off to different plateau values, depending on the PAA2K concentration. Three plateaus were obtained. First, in the control sample (without PAA2K), the pH value increased as the crystallization of calcium carbonate proceeded. After 30 h, the pH reached a plateau of about 8.0. In the presence of PAA2K, the pH values leveled off to different plateau values, depending on the PAA2K concentrations. At low (12) Zhang, S.; Gonsalves, K. E. Mater. Sci. Eng. 1995, C3, 117-124.

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with Ca2+ ions; consequently, local saturation in the chitosan-film vicinity was low, and the condition for forming the heterogeneous critical particles could not be satisfied. Even if heterogeneous nuclei can form at 1000 ppm PAA2K, crystal growth was inhibited for lack of sufficient Ca2+ in the chitosan-film vicinity. The SEM of calcium carbonate crystals growing on a chitosan thick film surface, from the supersaturated solution at different concentrations of PAA2K, are shown in Figure 9. In the control sample (without PAA2K), homogeneous nucleation occurred in the supersaturated calcium carbonate solution as carbon dioxide was released from the solution. The calcium carbonate crystals appeared as a multilayer precipitate on the chitosan film, randomly and sporadically. The crystals also occurred at the air-water interface as well as the inner edge of the polystyrene container in the same formation. In the presence of PAA2K, calcium carbonate crystals heterogeneously nucleated and grew only on the chitosan-film surface and covered the whole film. The crystals lost their well-developed edges and showed spherical symmetry around the center of nucleation. The spherical crystals were made up of a number of small rounded crystals. Figure 9. SEM photographs of calcium carbonate crystals growing on chitosan-film surfaces. Scale bar ) 10 µm.

concentrations, such as 100 and 200 ppm (w/w), pH values increased as fast as in the case without PAA2K in the first 30 h. After that the pH values increased rapidly and leveled off to a plateau of about 9.25. At a concentration of 600 ppm, crystallite nucleation and growth were inhibited at the beginning, resulting in a lower pH value than that of the lower PAA2K concentrations. Eventually, the pH value went up to 9.25. At a concentration of 2000 ppm, crystallization was totally inhibited, and the pH value remained at 6.00. No crystal was observed on the chitosan-film surface by SEM. In the cases of 1000 and 1500 ppm of PAA2K, the values fell to between 600 and 2000 ppm. At low concentrations of PAA2K, such as from 100 up to 600 ppm, the crystal habits and morphologies were the same. However, the crystal habits and morphologies were different from those at high concentrations. Since the dissociation constant of PAA is 4.5, the acid groups of PAA are mostly in the form of -COO- carboxylic ions at a pH range of 6.00-9.25. At higher concentrations (greater than 600 ppm) of PAA2K, the absorbed PAA2K on the chitosan film reached equilibrium after 600 ppm, resulting in more mobile PPA2K chains in solution. These mobile PAA2K chains in solution not only suppressed the homogeneous nucleation but also inhibited the heterogeneous nucleation and crystal growth on the chitosanfilm surface. This was attributed to the mobile PAA2K chains and adsorbed PAA2K chains on the chitosan film competing to attract Ca2+ ions. When the concentration of PAA2K was high, the mobile PAA2K chains binded

Conclusions PAA (MW ) 2000) proved to be an effective additive, promoting calcium carbonate heterogeneous nucleation on chitosan-film surfaces and suppressing homogeneous nucleation in solution. The adsorption of PAA2K on chitosan film revealed that PAA2K not only adsorbs on the chitosan-film surface but also penetrates into the film because of the short chain length of PAA2K and the swelling characteristics of chitosan films in aqueous solution. Protonated nitrogen cations and carboxylic anions were observed in the area where PAA2K was adsorbed on the chitosan film. Consequently, a high local saturation was obtained on the chitosan film. Nucleation was initiated from these charges and promoted in the high saturation area, and an interfacial layer between the chitosan film and calcium carbonate crystals was formed. The promotion of heterogeneous nucleation by PAA2K was concentration-dependent. At low concentrations of the PAA, heterogeneous nucleation and crystallization occurred and crystals of spherical morphology covered the entire film. At higher concentrations, even though nucleation did occur, crystallization of calcium carbonate on the chitosan film and in the solution was inhibited by mobile carboxylic anions of PAA in the solution. Local supersaturation around the chitosan film, a crucial requirement for heterogeneous nucleation of the film, could not be achieved at high PAA2K concentration because of the high affinity of mobile PAA2K chains for calcium ions in solution. LA970962S